The objectives of this research were to prepare ribavirin niosomes and evaluate the influence of niosomal encapsulation on drug liver targeting in rats. Ribavirin niosomes were prepared by the thin film hydration method using span 60, cholesterol, and dicetyl phosphate in molar ratios of (1:1:0), (4:2:0), (1:1:0.1), and (4:2:1). The prepared niosomes were characterized in vitro for vesicle size, drug entrapment, drug release profiles, and vesicular stability at refrigerator temperature. The results indicated that niosomes of the molar ratio (4:2:1) had a significantly (p < 0.05) higher entrapment percentage of ribavirin than the other molar ratios, moreover, they revealed sustained release characteristics as well as longer release pattern than other niosomal formulations. Accordingly, niosomes of molar ratio (4:2:1) was selected for in vivo liver targeting study. Separately, niosomal ribavirin dispersion and free ribavirin solution were administered as a single dose of 30 mg/kg by intraperitoneal injection into two groups of rats to compare the liver ribavirin concentration. The obtained results show that the niosomal formulation significantly increased ribavirin liver concentration (6-fold) in comparison with ribavirin-free solution. Based on the previous results, the use of niosomes as a drug delivery system for ribavirin has significant liver targeting properties, this is expected to improve the efficacy of low doses of ribavirin and minimize its toxic side-effects at higher doses.